Chemical sequencing of restriction fragments 3'-end-labeled with [35S]dATPalphaS

Although the phosphorous radioisotope 32P is routinely used in Maxam-Gilbert sequencing, it presents disadvantages that adversely affect safety and ease of use, resolution, and DNA degradation rates. Here, we introduce a new protocol utilizing the alternative isotope 35S for 3'-end-labeling DNA restriction fragments. In our method, plasmid DNA is labeled with [35S]dATPalphaS and 7 Sequenase Version 2.0. We have shown that bands on Maxam-Gilbert sequencing gels are sharp with extremely low background. In addition, a single labeling reaction produces DNA sufficient for 80 sequencing lanes, and the labeled DNA can be utilized for prolonged periods of time without significant degradation. We have further demonstrated the utility of our 35S-end-labeling procedure by successfully mapping the sequence-specificity of DNA damage induced by photoexcited riboflavin. Overall, we have shown that 35S can be used as a safe and practical alternative to 32P in the 3'-end-labeling of DNA restriction fragments.